Methods and apparatus for separation of particles

ABSTRACT

The present invention relates to a method for separating particles. The invention has particular advantages in connection with separating and purifying progenitor cells or stem cells obtained from bone marrow. The method comprises removing a desired volume of stem cell staring product from a donor/patient and eluting off a first contaminating cell type in a fluid chamber to create an enriched stem cell product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims priority from U.S. provisional patentapplication 60/521,552, filed May 21, 2004 and is a continuation-in-partof U.S. regular application Ser. No. 10/310,528, filed Dec. 4, 2002,which claims priority of U.S. provisional application 60/338,938, filedDec. 5, 2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for separating particles. Theinvention has particular advantages in connection with separating andpurifying progenitor cells or stem cells obtained from bone marrow.

2. Description of the Related Art

Bone marrow transplants are used to treat diseases once thoughtincurable. Diseases such as leukemia, aplastic anemia, Hodgkin'slymphoma, multiple myeloma, immune deficiency disorders and some solidtumors such as breast and ovarian cancers have been successfully treatedby bone marrow transplants.

Bone marrow is a spongy tissue found inside bones. The majority of thebone marrow is found in the breast bone, skull, hips, ribs and spine,and contain stem cells or progenitor cells which produce the body'sblood cells as well as other types of cells.

A stem cell/progenitor cell is characterized by having the ability toboth self-renew and differentiate into functionally distinct lineages.The differentiation pathway of a stem cell is unidirectional; that is,once committed to a particular cell lineage, the cell develops into aterminally differentiated cell. Stem cells are directed toward aparticular lineage by exposure to growth factors and their receptors.

Besides bone marrow, progenitor cells/stem cells are also found in someadult organs and tissues. These stem cells are known as adult stem cells(ASC). Stem cells are also found in embryos during early stages ofdevelopment and in fetal tissue, as well as in the umbilical chord.These stem cells are known as embryonic stem cells (ESC).

Until recently, it was believed that adult stem cell differentiation wasrestricted to the tissue in which the stem cell resides. Two examplesare hematopoietic stem cells that generate blood cells and oval cells(liver progenator cells), which generate hepatocytes.

Recently however, the concept of adult stem cells being only restrictedto their own tissue has been challenged by numerous reports that adultstem cells can jump lineages barriers and differentiate into cellsoutside their own tissue, in a process called stem celltransdifferentiation. These reports have revealed that stromal cellsobtained from adult bone marrow have many characteristics of mesenchymalstem cells. Pluripotent progenitor stromal cells may differentiate intovarious types of cells, including bone, muscle, fat, tendon orcartilage. Because of these recent findings, a process to obtain largeamounts of stem cells or progenitor cells to differentiate into variouscell types would be highly desirable.

Adult stem cells are present in bone marrow, blood, skin, muscle, liver,adipose tissue and brain. However, the frequency of stem cells in thesetissues is relatively low. For example, mesenchymal stem cell frequencyin bone marrow is estimated at between 1 in 100,00 and 1 in 1,000,000nucleated cells. Similarly, extraction of stem cells from tissueinvolves a complicated series of cell culture steps over several weeks.Any proposed clinical application using adult stem cells requires a highnumber of cells, high purity and external manipulation of cellularmaturation by processes of cell purification and cell culture.

Currently, purification of stem cells from bone marrow aspirate is doneusing Ficoll-Paque and Percol density gradients. Such methods ofpurification are problematic for several reasons. Firstly, suchpurifications are done manually by a technician. Although theseseparations are done under sterile conditions using laminar flow hoodsand the like, this method of purification does not occur in a closedsystem, which increases the risk of contaminating the cells withmicroorganisms. Secondly, ficoll and percol are chemicals, which must beremoved before the purified product may be given to a patient. Thirdly,in such separations, cells are lost during each step of the procedure.As discussed above, if the number of desired cells in a bone marrowaspirate is not high to begin with, every cell lost due to processingissues is critical to the end process.

Recent studies examining the therapeutic effects of bone-marrow derivedprogenitor/stem cells have used essentially the whole bone marrow toavoid the problems of cell purification. However, this creates otherproblems. Firstly, if bone marrow is injected directly into a damagedorgan, only a small percentage of stem cells are actually delivered tothe organ. As discussed above, the majority of bone marrow aspiratecontains other cells such as red blood cells and platelets. Secondly,there is a limited volume of cells which may be injected into an organ.It would be better therefore to maximize the amount of stem cellsdelivered to an organ without the problems associated with manualpurification.

In studies using animal models, it has been shown that unfractionatedmixtures of hematopoetic mononuclear cells that include differentiatedcells as well as progenitor stem cells, become incorporated intocollateral vessels.

The same principles used above in the animal studies are also being usedto treat humans. In patients who have suffered myocardial infarctions,loss of cardiac myocytes may lead to regional contractile dysfunction,and necrotized cardiomyocytes in infarcted ventricular tissues areprogressively replaced by fibroblasts to form scar tissue. Recentstudies have shown that transplanted fetal cardiomyocytes are able tosurvive in the damaged heart tissue and the transplanted cells limitedscar expansion and prevented postinfarction heart failure. Suchtreatment is not currently available due to current ethical and legalconsiderations. However, based on the results from the studies describedbelow, stem cells taken from adult bone marrow may potentiallysubstitute for fetal cardiomyocytes in this type of treatment.

In a clinical trial by Tateishi-Yuyama, autologous bone marrowmononuclear cells were injected into patients with ischemic peripheralvascular disease. Bone marrow cells were collected under generalanesthesia and injected into the gastrocnemius muscle of the ischemicleg in multiple sites. After treatment, significant improvement was seenin the ankle-brachial index (ABI), transcutaneous oxygen pressure andpain-free walking.

In another recent clinical trial, Hung-Fat Tse et al injected autologousbone marrow mononuclear cells into ischemic myocardium. The ischemicarea was injected intramyocardially with a mixture of CD34⁺, CD3⁺ Tcells and granulocytes. Following treatment, the number of anginalepisodes and nitroglycerin tablet usage decreased. Postinjection cardiacMRI demonstrated improved wall motion and thickness.

In one preliminary study done to date, one 50 mL aspiration of bonemarrow from patients who suffered an acute myocardial infarction wasaspirated from the iliac crest and immediately injected into the damagedarea of the heart. Repair of the damaged cardiac muscle and improvedcardiac function was seen.

An approximate volume of around 20 mL of bone marrow cells appears to bethe upper volume limit that can be injected into the heart. It may besurmised that cardiac repair and function may increase exponentially ifa greater volume of stem cells were collected either through multiplesticks or a greater aspiration volume and then concentrated into asmaller volume.

The present invention is directed towards avoiding the problemsassociated with manual purification of stem cells and towards the goalof purifying and concentrating large amounts of stem cells to be used intreating humans.

SUMMARY OF THE INVENTION

This invention includes a method for enriching stem cells, whichincludes the steps of removing a desired volume of stem cell startingproduct from a donor/patient to obtain a stem cell starting product,loading the stem cell starting product into a fluid chamber, flowing alow density fluid to the loaded stem cell starting product in the fluidchamber, centrifuging the fluid chamber; and eluting off a firstcontaminating cell type from the stem cell starting product in the fluidchamber to create an enriched stem cell product.

The method may further include a step of debulking the stem cellstarting product to remove a first contaminating cell type.

In a further aspect the invention relates to a method of concentratingthe enriched stem cell product.

It is another aspect of the present invention to treat a damaged organwith stem cells, which were collected from bone marrow and enriched andconcentrated using the above method.

Although the present invention is particularly directed to separatingstem cells or progenitor cells from other cells contained within a bonemarrow aspirate, it is understood that the techniques of the presentinvention can also apply to stem cells collected using other well knowncollection methods and from sources other than bone marrow aspirate,including, but not limited to, peripheral blood and umbilical cordblood. Therefore, both the foregoing general description and thefollowing detailed description are exemplary, and are intended toprovide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the invention and are incorporated in and constitute apart of this specification. The drawings illustrate an embodiment of theinvention and, together with the description, serve to explain theprinciples of the invention. In the drawings,

FIG. 1 is a perspective view of a disposable which could be used in theclosed system.

FIG. 2 is a perspective view of a closed system disposable containing afluid chamber, concentrator and separation vessel mounted on acentrifuge rotor.

FIG. 3 is a table showing elutriation results from the stem cellenrichment protocol of the present invention.

FIGS. 4 a-f are graphs of the elutriation results from FIG. 3 above.

DESCRIPTION OF THE PREFERRED EMBODIMENT

In the present invention, bone marrow is taken from a donor or patientusing any means known in the art. Typically, bone marrow is removed fromthe iliac crest of the donor/patient's pelvis via syringe draw. Atypical bone marrow harvest for hematopoetic reconstitution yieldsaround 2×10⁸ nucleated cells/kg body weight of the recipient. To obtainthe necessary amount of cells, it is usually required to remove around 1L of bone marrow. Anywhere between 0.1-25 mL of bone marrow may beaspirated from the bone with any one draw. Multiple aspirations aretypically necessary to obtain the desired amount of cells. If multipleaspirations are collected, they may be combined into a single source bagto provide a single source of stem cell starting product collected frommultiple syringe draws, or may be collected into multiple source bags,each containing stem cell starting product collected from a singlesyringe draw. The single source bags may be processed individually, ormay be combined either before or after processing.

Stem cells may also be separated from peripheral blood. A COBE® SPECTRA™blood component centrifuge manufactured by Gambro BCT, Inc. of Coloradomay be used to initially separate blood into components. Stem cells aretypically found in the white blood cell fraction. The separated cellfraction containing white blood cells and stem cells may then be used asthe stem cell starting product in the enrichment procedure describedbelow.

Stem cells are also found in umbilical cord blood. The proceduredescribed below may also be used to enrich stem cells from cord blood.

One way to enrich a specific subset of cells from a fluid containingmany cell types is to use elutriation technology. Elutriation could beused to separate progenitor/stem cells from other cells contained inbone marrow, peripheral blood or umbilical cord blood. The enrichedproduct may then be concentrated to a final volume appropriate for thedesired application.

In one common form of elutriation, a cell batch such as the stem cellstarting product collected in the source bag/bags is introduced into afunnel-shaped chamber located in a spinning centrifuge. A flow of liquidelutriation buffer is then introduced into the chamber containing thecell batch. As the flow rate of the liquid buffer solution is increasedthrough the chamber (usually in a stepwise manner), the liquid sweepssmaller sized, slower-sedimenting cells toward an elutriation boundarywithin the chamber, while larger, faster-sedimenting cells migrate to anarea of the chamber where the centrifugal force and the sedimentation(drag) forces are balanced.

Thus, centrifugal elutriation separates particles having differentsedimentation velocities. Stoke's law describes sedimentation velocity(SV) of a spherical particle, as follows:${SV} = {\frac{2}{9}\frac{{r^{2}( {\rho_{p} - \rho_{m}} )}g}{\eta}}$where,

-   -   r is the radius of the particle,    -   ρ_(p) is the density of the particle,    -   ρ_(m) is the density of the liquid medium,    -   η is the viscosity of the medium, and    -   g is the gravitational or centrifugal acceleration.

Because the radius of a particle is raised to the second power in theStoke's equation and the density of the particle is not directly relatedto the size of a cell, its density greatly influences its sedimentationrate. This explains why larger particles/cells generally remain in achamber during centrifugal elutriation, while smaller particles/cellsare released, if the particles have similar densities.

Specific cell subsets to date have initially been separated from, ordebulked of, red blood cells by density gradient centrifugation, usingvarious separation media. In density gradient centrifugation, a sampleis layered on top of a media support and centrifuged. Under centrifugalforce, the particles in the sample will sediment through the media inseparate zones according to their density. As discussed above, manualdensity gradient separation is not done in a closed system and requiresboth a contamination free environment and chemical gradients, both ofwhich are undesirable.

It is known that red blood cells under proper conditions have thetendency to adhere to each other forming red blood cell rouleaux.Rouleaux formation and size, and therefore red cell sedimentationvelocity, is influenced by the hematocrit of the cell suspension,exposure to shear, protein concentration, and presence of sedimentationagents.

Reference will now be made in detail to the embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. Wherever possible, the same reference numbers are used in thedrawings and the description to refer to the same or like parts.

The COBE® SPECTRA™ centrifuge incorporates a one-omega/two-omegasealless tubing connection as disclosed in U.S. Pat. No. 4,425,112 toIto, the entire disclosure of which is incorporated herein by reference.Although the embodiments of the invention are described in combinationwith the COBE® SPECTRA™ centrifuge, this reference is made for exemplarypurposes only and is not intended to limit the invention in any sense.

As will be apparent to one having skill in the art, the presentinvention may be advantageously used in a variety of centrifuge devicescommonly used to separate cell subsets into desired cell types. Inparticular, the present invention may be used with any centrifugalapparatus regardless of whether or not the apparatus employs aone-omega/two-omega sealless tubing connection.

As embodied herein and illustrated in FIG. 1, the present inventionincludes a particle separation disposable system 10 for use with acentrifuge rotor 12. Preferably, the centrifuge rotor 12 is coupled to amotor (not shown) via an arm 14, shown in FIG. 2, so that the centrifugerotor 12 rotates about its axis of rotation A-A.

As shown in FIG. 2, a holder 16 is provided on a top surface of therotor 12. The holder 16 releasably holds a fluid chamber 18 on the rotor12 such that an outlet 20 for cells other than red blood cells,hereinafter called the outlet of the fluid chamber 18, is positionedcloser to the axis of rotation A-A than the inlet 22 of the fluidchamber 18. The holder 16 preferably orients the fluid chamber 18 on therotor 12 with a longitudinal axis of the fluid chamber 18 in a planetransverse to the rotor's axis of rotation A-A. In addition, the holder16 is preferably arranged to hold the fluid chamber 18 on the rotor 12with the fluid chamber outlet 20 for cells other than red blood cellsfacing the axis of rotation A-A. Although the holder 16 retains thefluid chamber 18 on a top surface of the rotor 12, the fluid chamber 18may also be secured to the rotor 12 at alternate locations, such asbeneath the top surface of the rotor 12. It is also understood that thefluid chamber 18 could be secured by other well known fixative devicesor by other methods other than the holder as shown.

The fluid chamber 18 has smooth sides as shown in FIGS. 1 and 2 asdescribed below. As shown in FIGS. 1 and 2, the inlet 22 and outlet 20of the fluid chamber 18 are arranged along a longitudinal axis of thefluid chamber 18. A wall 21 of the fluid chamber 18 extends between theinlet 22 and outlet 20 thereby defining inlet 22, the outlet 20, theside and an interior of the fluid chamber 18.

The fluid chamber 18 includes two frustoconical shaped sections 25, 27joined together at a maximum cross-sectional area 23 of the fluidchamber 18. The interior of the fluid chamber 18 tapers (decreases incross-section) from the maximum cross-sectional area 23 in oppositedirections toward the inlet 22 and the outlet 20. Although the fluidchamber 18 is depicted with two sections (25, 27) having frustoconicalinterior shapes, the interior of each section may be paraboloidal, or ofany other shape having a major cross-sectional area greater than theinlet or outlet area.

The fluid chamber 18 may be constructed from a unitary piece of plasticor from separate pieces joined together using known fixative or sealingmethods to form separate sections of the fluid chamber 18. The fluidchamber 18 may be formed of a transparent or translucent copolyesterplastic, such as PETG, to allow viewing of the contents within thechamber interior with the aid of an optional strobe (not shown) during aseparation or debulking procedure.

As shown in FIG. 1, the system 10 which depicts a closed systemdisposable further includes a first conduit or line 28, second or debulkconduit or line 30, an inlet conduit or line 32 in fluid communicationwith the inlet 22 of the fluid chamber 18, and a three-way or Yconnector 34 having three legs for flow or fluidly connecting the firstconduit 28, second or debulk conduit 30, and inlet line 32. The firstconduit 28 includes a coupling 36 for flow-connecting the first conduit28 with conduit or line 27, coupling 39 and the single (or multiple)source bag/s 38 containing stem cell starting product to be separatedinto stem cells and other cells. Likewise, the first conduit 28 isconnected by coupling 36 to conduit or line 37 which includes couplings40 for flow-connecting the first conduit 28 with a second source 42containing a low density diluting, sedimentation or elutriation fluid.An in-line filter 3 may or may not be placed within conduit 37 to filterfluid from source 42. The couplings 36, 39 and 40 are preferably anytype of common medical coupling devices, such as spikes or steriletubing connectors.

As shown in FIG. 1, the first conduit 28 includes a first tubing loop44. During use, the first tubing loop 44 is mounted in a peristalticpump (not shown) for respectively pumping the stem cell starting productto be separated and the diluting, sedimentation or elutriation fluidfrom the first and second sources 38 and 42, respectively.

The stem cell starting product from the first source bag 38 and thediluting, sedimentation or elutriation fluid from the second source 42flow through the respective first conduit 28 to the three-way connector34. These substances then flow through the inlet line 32 into the inlet22 of the fluid chamber 18. In the fluid chamber 18, turning with rotor12, the cells in the bone marrow in the centrifugal field separateaccording to differences in sedimentation velocity leaving fastersedimenting cells in the fluid chamber 18 and allowing some slowersedimenting cells to flow from the fluid chamber 18 as will be describedbelow.

As the fluid chamber 18 is loaded with stem cell starting product as ismore fully described below, the fluid and cells having a relativelyslower sedimentation velocity, which generally includes white bloodcells and stem cells, will flow through the fluid chamber outlet 20 intoconduit tubing or line 48. As shown in FIG. 2, the tubing 48 mayoptionally be connected to an inlet 50 of a separation vessel 52 oroptional cellular concentrator mounted to the centrifuge rotor 12.

If an optional concentrator is used, it will be placed adjacent to anouter portion of the centrifuge rotor 12. The concentrator 52 has acollection well 54 for collecting particles flowing into theconcentrator 52. Rotation of centrifuge rotor 12 sediments particlesinto the collection well 54, while slower sedimenting fluid and possiblysome slower sedimenting particles remain above a top boundary of thecollection well 54. The collected particles in the collection well 54can include any cells or particles that have exited the fluid chamber18, or separated subsets of white blood cells and stem cells, as notedabove.

In the embodiment shown in FIG. 2, the optional concentrator 52 isplaced in a groove 64 formed in the rotor 12. Preferably, theconcentrator 52 is a channel formed of a semi-rigid material so that avalley 66 in an outer wall of the groove 64 forms the collection well 54when the concentrator 52 expands in response to fluid and particles inthe concentrator 52 encountering centrifugal forces. As shown in FIG. 2,the top surface of the rotor 12 preferably includes retainer grooves forreceiving the first and second conduits 28 and 30, three-way connector34, inlet line 32, tubing 48, particle concentrate line 58, and fluidoutlet line 62. If a tubing set without a concentrator is used, such asshown in FIG. 1, the rotor will not have groove 64 or valley 66.

As shown in FIG. 1, the fluid outlet line 62 is fluidly coupled at oneend to outlet 20 and at the other end to a fluid collection container 61for collecting fluid removed from the fluid chamber 18, and the particleconcentrate line 58 is fluidly coupled to one or more particlecollection containers 70 for collecting particles removed from the fluidchamber 18. Although only one particle collection container 70 is shown,it should be appreciated that as many particle containers as needed tocollect elutriation fractions may be used. For example, if twelvefractions (such as shown in FIG. 3) are collected, each fraction may becollected in a separate collection container. Therefore, twelvecollection containers 70 would be attached to particle concentrate line58.

Preferably, the particle concentrate line 58 includes a tubing loop 72capable of being mounted in a peristaltic pump for pumping particlesthrough the particle concentrate line 58. The pump for tubing loop 72regulates the flow rate and concentration of particles in particleconcentrate line 58. The stem cells will be collected into bag 70. It isunderstood that any number of bags 70 can be used to collect the desiredsubsets of stem cells. Platelets, which are considered to becontaminating cells in a stem cell enrichment procedure such asdescribed here, can also be collected in a separate bag if desired.

After sedimentation in chamber 18, as is more fully described below, redblood cells, which are considered to be contaminating cells in a stemcell enrichment procedure, are removed through inlet 22 to inlet conduit32. The debulked red blood cells then pass through Y connector 34 todebulking conduit 30. As shown in FIG. 1, conduit 30 is fluidly coupledto a red blood cell collection container or debulked cell collectioncontainer 31 for collecting red blood cells collected during thedebulking procedure. Preferably the red blood cell collection or debulkline or conduit 30 includes a tubing loop 46 capable of being mounted ina peristaltic pump for pumping red blood cells through conduit 30.

To control flow rates of substances and rotational speed of the rotor 12during operation of the system 10, a controller (not shown) controlspumps (not shown) for pumping substances through the tubing loops 44, 46and 72 and controls a motor (not shown) for rotating the centrifugerotor 12.

A preferred method of separating components of blood and, in particular,separating stem cells and white blood cells from red blood cells isdiscussed below with reference to FIGS. 1-4. Although the invention isdescribed in connection with a blood component separation process andspecifically a stem cell separation or fractionation process, it shouldbe understood that the invention in its broadest sense is not solimited.

Initially, bone marrow aspirate is drawn from a patient using a syringe2 and needle 4. Depending upon the number of stem cells desired, bonemarrow may be collected from a donor/patient in very small volumes ofaround 0.1 mL, up to larger volumes of around 25 mL, using one or moreneedle sticks. This bone marrow aspirate will henceforth be known as thestem cell starting product regardless of the way it was collected from adonor/patient. It should be noted that the larger the amount of bonemarrow removed from a donor/patient in a single draw, the morecontaminated the sample may be with other components of bone marrow suchas red blood cells and platelets, and the more separation and enrichmentwill be required. Small aspirates (around 0.2 mL) will be lesscontaminated with platelets and red blood cells than larger volumes. Theaspirates may be injected into a storage bag (not shown) or may beinjected directly into source bag 38 as shown in FIG. 1.

Filtration of contaminating bone fragments and other solid material maybe necessary before the elutriation procedure. Any filters 6 known inthe art may be used. The filter may be placed anywhere within the closedsystem so long as it is placed before the elutriation chamber 18. Asexamples, not meant to be limiting, the filter may be connected anywherewithin the tubing line leading to source bag 38 (as shown in FIG. 1).The bone marrow aspirate may gravity drain through tubing 8, throughfilter 6 and into source bag 38. The filter may also be placed directlyon the end of the syringe used to aspirate the bone marrow. The bonemarrow is forced through the filter into source bag 38 by application ofdownward pressure to the syringe. The filter may also be placed withinthe tubing line 10 leading out of source bag 38.

The stem cell starting product is placed in the first source 38 shown inFIG. 1, and the first source 38 is coupled to the first conduit 28through conduit 27. In addition, the second source 42 containing thediluting, sedimentation or elutriation fluid is coupled to the conduit28 through the conduit 37. The centrifuge rotor 12 is rotated about theaxis of rotation A-A (see FIG. 2), at approximately 2400 rpm. The stemcell starting product is pumped from source 38 at a low flow rate andloaded into the fluid chamber 18. The flow of stem cell starting productfrom source 38 is then stopped by a valve or other well-known mechanism.Flow of diluting, sedimentation or elutriation fluid is then started torinse conduit 28 and/or wash the loaded stem cell starting product.Small particles (such as platelets) may be removed from conduit 28simply by the flow of the fluid during this flowing step. The diluting,sedimentation fluid or elutriation fluid passes through conduit 28 and Yconnector 34, and inlet conduit 32 into the inlet 22 of chamber 18.

The inlet pump 44 associated with the tubing loop is stopped to stop theflow of low density diluting, sedimentation or elutriation fluid intothe chamber 18. As the centrifuge continues to rotate, the stem cellstarting product loaded in the chamber sediment under the resultingcentrifugal force.

After sedimentation of the particle constituents of the stem cellstarting product, the pump associated with tubing loop 46 is activatedto remove or debulk at a low flow rate the sedimented red blood cellsthrough the inlet 22 of the chamber 18 and then through inlet conduit 32and debulking conduit 30 to container 31.

After removal of red blood cells, the stem cells and white blood cellsremaining in chamber 18 can be further separated as described below, orthe inlet pump associated with tubing loop 44 can be restarted toreintroduce a second batch of blood product from source 38 into chamber18. This would be desirable if multiple bone marrow aspirations weredone.

The elutriating step for separating stem cells and white blood cellsinto the desired subsets can be done after each debulking procedure orafter the source 38 is empty of stem cell starting product. The onlyrequirement is that there be a sufficient number of stem cells and whiteblood cells in chamber 18 to achieve effective separation orfractionation. Therefore, the white blood cell and stem cell content ofthe stem cell starting product should be considered in determining thesequence order of the elutriation step.

For collection of fractionated or separated white blood cells or stemcells, an operator, after debulking or after the first source 38 isempty, slowly increases the inlet pump speed associated with tubing loop44, decreases the centrifuge speed, or increases the density orviscosity of the diluting, sedimentation or elutriation fluid toseparate the cells in chamber 18 into subsets by elutriation, as is wellknown in the art. Such separated subsets may then be concentrated in theoptional concentrator 52 (if used), or simply be removed to bag/s 70.

Although the preferred embodiment discloses separating the white bloodcells and stem cells in subsets using elutriation in chamber 18, it isalso understood that a second separate chamber (not shown, but similarto chamber 18) could be fluidly connected between chamber 18 andoptional concentrator 52 (if one is used) wherein the white blood cellsand stem cells can be further separated into subsets or concentratedusing the elutriation separation process in the second chamber. Also,the elutriative separation can occur after the white blood cells andstem cells are collected into a bag 70 as a separate processing step.

The loading, flowing of low density fluid, sedimenting, debulking andelutriating steps, (if done after debulking), described above may berepeated until the entire stem cell starting product from one or moreaspirations has been separated or enriched into desired components ordesired subsets and debulked of red blood cells. Alternatively, asmentioned above, the loading, flowing of low density fluid, sedimentingand debulking steps may be repeated multiple times until the entire stemcell starting product has been debulked of red blood cells. The entiredebulked stem cell product may than be elutriated in one elutriationstep.

It is understood that the protein and sedimentation agents used to formthe diluting, sedimentation fluid could be any fluid known in the art.It is also understood that the low density fluid could be media orplasma.

Although the diluting, sedimentation or elutriation fluid is added onlyat certain parts of the process, it is understood that otherconfigurations are possible. For example, the fluid chamber 18 could bemodified to include separate inlets for blood components and diluting orsedimentation fluid. The diluting or sedimentation fluid could also beadded to the blood components in the first source 38 before, or at thebeginning of, a batch separation process. It is further understood thatthe selection of elutriation fluid may depend on whether the subsetswill be separated by an elutriation technique after debulking.

As the stem cell starting product is being loaded into the separationchamber 18 and during the elutriating step, the diluting, sedimentationor elutriation fluid, plasma, platelets, and the white blood cells andstem cells flow from the fluid chamber outlet 20 through the particlecollect line 58 to the collect bag/s 70, while the diluting fluid andplasma flow through the fluid outlet 60 and fluid outlet line 62 tocontainer 61. This separates the platelets and other particles from thediluting fluid and plasma.

The instant debulking procedure could achieve effective removal of RBCswithout a significant loss of stem cells, and can achieve such in aclosed system. The capacity of the system of the instant invention canbe increased by placing several small chambers in parallel or in series,or by using one large chamber. Ideally, either the combined chambers ora single chamber should be capable of debulking and/or elutriatingbetween approximately 10 to 150 ml of stem cell starting product. Thecurrent disposable could easily be adapted to accommodate multiplechambers or one large chamber, provided the chamber could be recessed inthe rotor 12.

The disposable particle separation system may also optimally includesensors at various output locations such as in the particle concentrateline for monitoring the types of cells and concentration beingcollected. Any known type of a sensor could be used.

EXAMPLES Example 1

In the following experiment, stem cells were mobilized from bone marrowinto the peripheral blood and an apheresis sample was collected usingSpectra.

The elutriation protocol used to enrich stem cells from the startingstem cell product is set out in the table below. The columns set out theflow rate (mL/min), rotor speed and volume of elution fluid used to flowthrough the fluid chamber to enrich stem cells and white blood cellsfrom peripheral blood. This procedure may also be used to debulk andenrich stem cell aspirated from bone marrow. The volume of elution fluidused was 500 mL. The rotor speed was maintained at 2400 rpm, except forthe last fraction, which was collected with the rotor off. Twelvefractions were eluted and collected as well as a pre-fraction, which wascollected before the elutriation procedure was begun. Fraction ml/minrotor volume Pre na na 500 ml 1 37 2400 500 ml 2 77 2400 500 ml 3 812400 500 ml 4 85 2400 500 ml 5 90 2400 500 ml 6 95 2400 500 ml 7 1002400 500 ml 8 105 2400 500 ml 9 110 2400 500 ml 10 115 2400 500 ml 11120 2400 500 ml 12 120 off 250 ml

In the experiments, the eluted fractions were analyzed using flowcytometry to count and classify blood cell types. Fluorescent antibodieswhich are specific to receptors on the surface of the cells were used asmarkers to measure the different cell types. CD45 is a marker for whiteblood cells, CD34 is a marker for stem cells, CD3 is a marker forT-cells, CD 14 is a marker for monocytes, and CD19 is a marker forB-cells. Traditional flow cytometry gating/counting methods were used.

The results are shown in the table of FIG. 3 below. The table shows thetotal number of each cell type which elutes off in each fraction.

The cell count data is also depicted graphically FIGS. 4 a-4 f. FIG. 4 ashows the elutriation profile of red blood cells. FIG. 4 b shows theelutriation profile of the general category of white blood cells, whichwill include all subsets of white blood cells as well as stem cells andother similarly sized cells. FIG. 4 c shows the elutriation profile ofstem cells. FIG. 4 d shows the elutriation profile of T-cells. FIG. 4 eshows the elutriation profile of monocytes, and FIG. 4 f shows theelutriation profile of B-cells.

The graphs show three white blood cell peaks: an early CD19 (B-cells)peak (FIG. 4 f) which overlaps with the RBC peak (FIG. 4 a); a mid-peakcontaining CD3 (T-cells) (FIG. 4 d); and a late peak containing CD34(stem cells) (FIG. 4 c), and CD14 (monocytes) (FIG. 4 e). Using theseresults, elutriation fractions which contain enriched fractions ofdifferent cell types may be selectively collected in bag/s 70. Forexample, if primarily stem cells were desired, fractions 9-12 should becollected. However, as can be seen from FIG. 3 e, monocytes will also becollected in this enriched stem cell fraction. A further processingstep, such as antibody specific adsorption as discussed above may bedesired. Alternatively, other contaminating cell types such as B cellsand T cells may be eluted off before the desired enriched fraction iscollected.

Example 2

The above-described method may be incorporated into a method forenriching progenitor cells from bone marrow aspirate. The enrichedprogenitor cells obtained by the described method may be furtherconcentrated into a smaller volume. Progenitor cells obtained by thismethod may be injected directly into damaged tissue to heal and re-growthe injured tissue.

Depending on the type of injured tissue to be treated, variable amountsof bone marrow may be collected. The bone marrow could be collected fromthe patient to be treated, or could be collected from a suitable donor.

The final volume and number of cells that will be concentrated willdepend on the type of organ to be treated. As one example, if it isdesired to treat cardiac muscle, the starting volume of the stem cellstarting product may be concentrated down to a volume of approximately20 mL, which may be the approximate maximum volume practical to inject(in one or more injections) into the heart. The injection/s may be giveneither intramuscularly or intravenously, or both.

An additional step may be to select for specific progenitor cell typesfrom the final enriched product. Such selection may be done by any meansknown in the art, but may include cell selection using antibodiesspecific to subtypes of progenitor cells such as mesenchymal stem cells,which could differentiate into different tissue types uponinjection/transplantation into the damaged organ, as but one example,not meant to be limiting.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the structure andmethodology of the present invention without departing from the scope orspirit of the invention. In view of the foregoing, it is intended thatthe present invention cover modifications and variations of thisinvention provided they fall within the scope of the following claimsand their equivalents.

1. A method of enriching stem cells comprising the steps of: removing adesired volume of stem cell starting product from a donor/patient toobtain a stem cell starting product; loading the stem cell startingproduct into a fluid chamber; flowing a low density fluid into theloaded stem cell starting product in the fluid chamber; centrifuging thefluid chamber; and eluting off a first contaminating cell type from thestem cell starting product in the fluid chamber to create an enrichedstem cell product; and collecting the enriched stem cell product.
 2. Themethod of claim 1 further comprising a debulking step to remove a firstcontaminating cell type from the stem cell starting product.
 3. Themethod of claim 2 wherein the debulking step occurs before the elutingstep.
 4. The method of claim 1 further comprising concentrating theenriched stem cell product.
 5. The method of claim 1 wherein the stemcell starting product further comprises bone marrow.
 6. The method ofclaim 1 wherein the stem cell product further comprises peripheralblood.
 7. The method of claim 1 wherein the stem cell product furthercomprises umbilical cord blood.
 8. The method of claim 5 wherein thestep of removing bone marrow further comprises filtering the bonemarrow.
 9. The method of claim 8 wherein the filtering step occursbefore the loading step.
 10. The method of claim 1 further comprisingeluting off a second contaminating cell type from the starting productin the fluid chamber.
 11. The method of claim 1 further comprisingeluting off a third contaminating cell type from the starting product inthe fluid chamber.
 12. The method of claim 1 further comprising elutingoff a fourth contaminating cell type from the starting product in thefluid chamber.
 13. The method of claim 1 comprising repeating theloading, flowing, centrifuging, eluting and collecting steps as manytimes as necessary to obtain the desired number of enriched stem cells.14. The method of claim 2 comprising repeating the loading, flowing,debulking, and centrifuging steps as many times as necessary to removeundesired cells before the eluting step.
 15. The method of claim 4further comprising repeating the concentrating step as many times asnecessary to obtain the desired number of enriched, concentrated stemcells.
 16. The method of claim 1 wherein the flowing step comprisesflowing a low density fluid into the fluid chamber at a flow rate ofaround 37 mL/min at a volume of around 500 mL.
 17. The method of claim 2wherein the first contaminating cell type is red blood cells.
 18. Themethod of claim 10 wherein the second contaminating cell type isplatelets.
 19. The method of claim 11 wherein the third contaminatingcell type is T-cells.
 20. The method of claim 12 wherein the fourthcontaminating cell type is B-cells.
 21. A method of treating a damagedorgan with stem cells collected from bone marrow comprising the stepsof: removing a desired volume of bone marrow from a donor/patient toobtain a bone marrow starting product; loading the bone marrow startingproduct into a fluid chamber; flowing a low density fluid into theloaded bone marrow starting product in the fluid chamber; centrifugingthe fluid chamber; debulking a first contaminating cell type from thebone marrow starting product in the fluid chamber; eluting off a secondcontaminating cell type from the bone marrow starting product in thefluid chamber to create an enriched stem cell product; concentrating theenriched stem cell product to obtain a concentrated, enriched stem cellproduct; and injecting the concentrated enriched stem cell product intothe damaged organ.
 22. The method of claim 21 further comprisingdetermining the amount of stem cells needed to treat the damaged organ.23. The method of claim 21 wherein the step of injecting furthercomprises injecting the concentrated enriched sample into cardiactissue.
 24. The method of claim 21 wherein the step of injecting furthercomprises injecting the concentrated enriched stem cell productintravenously.
 25. The method of claim 21 wherein the step of injectingfurther comprises injecting the concentrated enriched stem cell productintramuscularly.
 26. The method of claim 21 wherein the step ofinjecting further comprises injecting the concentrated enriched stemcell product both intravenously and intramuscularly.
 27. The method ofclaim 21 wherein the step of injecting further comprises injecting theconcentrated enriched stem cell product in multiple injections.
 28. Themethod of claim 21 wherein the step of injecting further comprisesinjecting the concentrated enriched stem cell product in a singleinjection.